Development in the cellular slime mold Dictyostelium discoideum is regulated in part by extracellular molecules that interact with cell surface receptors. This application is directed at understanding the molecular and biochemical mechanisms by which signal transduction pathways control both cellular morphogenesis and gene expression during the pre-aggregation and aggregation stages of development. Using molecular techniques, we have identified and cloned genes whose products are essential in controlling this and other stages of development. These include genes encoding four G alpha protein subunits, each with a distinct developmental pattern of expression, a developmentally regulated phosphotyrosine phosphatase, and developmentally regulated putative serine/threonine kinases. Gene disruptions and overexpression studies have indicated that the proteins encoded by each of these genes play essential roles in development. Our analysis indicates that G alpha2 couples to the cAMP receptor cARI and regulates diverse signal transduction pathways that mediate chemotaxis during aggregation and the induction of pulse-induced genes. In this application, we propose to dissect the signal transduction pathways regulated by these proteins using molecular techniques to produce appropriate strains that either do not express the proteins or express modified proteins. In vitro analysis will be used to characterize the effects of the mutations at a biochemical level. Using molecular complementation, we propose to isolate additional genes in the signaling pathways that have been previously identified by in vivo mutations. We will also continue to characterize the function of the cell-type-specific ras gene within the signal transduction pathway. In order to understand the molecular basis of receptor-mediated control of gene expression during aggregation, we plan to further identify the cis-acting regulatory regions of genes regulated by cAMP pulses and to purify the regulatory trans-acting factors that mediate the response at the level of transcription.